Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.
Known methods for analysing gene expression include the use of PCR or nested PCR to amplify a selected cDNA sequence, representing the expression product of a single gene from a pool of cDNA molecules representing many different genes. In a single-gene PCR reaction the gene expression products from a single gene are typically amplified using one pair of primers in a single round of PCR. In a typical nested PCR amplification reaction a single very rare sequence is amplified by two sequential PCR reactions, each consisting of 30-40 cycles using a single nested primer combination. Nested PCR is normally used to obtain a clone of a rare sequence and is not normally considered for the quantification of gene expression or identification of a gene. In order to quantify the level of gene expression using PCR, the amount of each PCR product of interest is typically estimated using conventional quantitative real-time PCR (qPCR) to measure the accumulation of PCR products in real time after each cycle of amplification. This typically utilises a detectible reporter such as an intercalating dye, minor groove binding dye or fluorogenic probe whereby the application of light excites the reporter to fluoresce and the resultant fluorescence is typically detected using a CCD camera or photomultiplier detection system, such as that disclosed in U.S. Pat. No. 6,713,297 which is hereby incorporated by reference. While single-gene analysis methods are now routine, difficulties arise when multiple gene expression products must be amplified within the same PCR reaction in a multiplex PCR. In a multiplex amplification reaction, each individual gene expression product must compete for reaction components during PCR, such that the products of highly expressed genes, which are present at a high copy number at the start of the PCR reaction, effectively prevent the amplification of low copy number gene products by sequestering vital reaction components. This results in a pool of amplified gene products that may represent only a small number of highly expressed genes. Analysis of these products is further complicated by the large variation that occurs between replicate experiments making the accurate quantification of gene expression very difficult. While optimisation of primers and reaction components can alleviate this problem to some degree, this typically involves extensive experimentation and becomes far more difficult as the number of genes to be analysed in a multiplex reaction is increased, whereby four genes would typically be the maximum number that could be reliably analysed in an extensively optimised system. Gene expression analysis typically requires that the amount of each gene expression be estimated, which is further complicated in a multiplex PCR.
Modern Approaches to Multiplex PCR
Modern approaches to this problem include the use of fluorogenic detection systems such as Taqman® probes to detect each gene expression product individually by binding to these and releasing a specific detectible fluorescence (Exner M. M., and Lewinski. M. A. (2002). Sensitivity of multiplex real-time PCR reactions, using the LightCycler and the ABI PRISM 7700 Sequence Detection System, is dependent on the concentration of the DNA polymerase (Molecular and Cellular Probes. October 2002;16(5):351-7). These probes and their uses are described in U.S. Pat. Nos. 5,210,015; 5,487,972; 5,804,375; 5,994,056; 5,538,848 and 6,030,787 and are hereby incorporated by reference. This requires a real time thermal cycling machine having multiple channels for detecting fluorescence at different wavelengths. In addition, Taqman® probes are expensive to purchase and may also limit the particular region of sequence that can be analysed due to specific sequence requirements for probe binding.
Other fluorogenic approaches include the use of generic detection systems such as SYBR-green dye, which fluoresces when intercalated with the amplified DNA from any gene expression product as disclosed in U.S. Pat. Nos. 5,436,134 and 5,658,751 which are hereby incorporated by reference. While SYBR-green is inexpensive to use and has excellent fluorogenic properties it is not normally appropriate for estimating the level of gene expression in a multiplex PCR as the source of the fluorescence, with regard to each gene product, cannot be reliably determined.
Irrespective of the use of fluorogenic probes or SYBR-green dye as the detection system, multiplex PCR still suffers from the same problem where gene expression products compete for reaction components, thereby hampering the accurate quantification of gene expression from multiple genes.
High Throughput Approach
An alternative approach to multiplex gene expression measurements includes the use of microarrays. Microarrays can be used to quantify the expression of thousands of genes simultaneously. However microarrays typically require extensive operator training, large amounts of sample RNA, and expensive equipment. In addition, while the number of genes that can be analysed is large, the resultant quantification of gene expression is far less accurate, often leading to false positives.
Thus there is a need for a simple and inexpensive method that is suitable for use in any setting where the accurate quantification of the expression of multiple genes is required or where the detection of specific nucleic acids is required or where the production of multiple nucleic acid products is required. The invention is particularly suited to the amplification and detection of nucleic acids from very small samples such as blood spots, laser dissection microscopy samples, single cells and samples containing partially fragmented nucleic acids such as those taken from aged samples and formalin-fixed paraffin-embedded (FFPE) sections. However, the methods of the invention are equally applicable to larger samples and also those of high quality. Examples of settings in which the invention could be useful include but are not limited to: diagnostics; prognostics; forensics; environmental and product testing and monitoring; biological weapons detection; research and the like.
It is therefore an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.